SUMO proteins are a conserved family of ubiquitin-related proteins that become covalently linked to other cellular proteins. The conjugation pathway for all SUMO proteins is similar to the ubiquitin conjugation pathway: SUMO proteins must be processed to yield a C-terminal di-glycine motif. After processing, the first step in the SUMO conjugation pathway is the ATP-dependent formation of a thioester bond between SUMO proteins and their activating (E1) enzyme. The second step is the formation of a thioester bond between SUMO proteins and their conjugating (E2) enzyme, Ubc9. In the last step, an isopeptide bond is formed between SUMO proteins and substrates through the cooperative action of Ubc9 and protein ligases (E3). There are three broadly expressed human SUMO paralogues, which have been implicated in a variety of cell functions, including nuclear trafficking, chromosome segregation, chromatin organization, transcription and RNA metabolism. SUMO-1 is about 45% identical to SUMO-2 and SUMO-3, which are 96% identical to each other. We are interested in the distinct roles of individual SUMO paralogues within vertebrate cells, as well as in the how these proteins are specifically recognized by enzymes involved in their conjugation and deconjugation from their targets. It is currently unclear whether SUMO-1, -2 and -3 function in ways that are unique, redundant or antagonistic. Moreover, all three paralogues share common E1 and E2 enzymes, while the specificity of SUMO ligases and proteases is not well understood. It has been difficult to address this question experimentally in the past, because superphysiological levels of individual SUMO proteins can cause a loss of paralogue specificity, and because the dynamics and localizations of these proteins can only be imprecisely estimated within fixed cells. To address the dynamic properties of SUMO paralogues, we have developed stable HeLa-derived cell lines that express biofluorescent SUMO chimeras at levels comparable to the endogenous proteins. Through live imaging and photobleaching studies, we have found that SUMO-1 differs from SUMO-2 and SUMO-3 in both it localization and its dynamics throughout the cell cycle. Additionally, we found significant differences between SUMO-1 dynamics in different subnuclear compartments. Our findings demonstrate that mammalian SUMO paralogues show discrete temporal and spatial patterns of utilization throughout the cell cycle, arguing that they are functionally distinct and specifically regulated in vivo. The linkage of SUMO proteins to their substrates can be severed by SUMO proteases, so it is likely that SUMO modification is highly dynamic in vivo. Both processing and deconjugation are mediated by the same family of SUMO proteases (SENP1, 2, 3, 5, 7 and SUSP1; collectively referred to as SENPs). Comparatively little is known, however, about the enzymatic differences and distinct biological roles of these enzymes. We have selectively suppressed the synthesis of individual SENPs in cell lines stably expressing different GFP-SUMO fusion proteins, using RNA interference (RNAi). We have observed that different GFP-SUMO fusions become re-localized after the depletion of individual SENPs in distinct, paralogue-specific patterns, suggesting that these enzymes show considerable in vivo specificity. Biochemical analysis supports the notion that SENPs show strong paralogue specificity. These findings argue that SENPs are highly specific, and that they play a critical role in determining the spectrum of SUMO conjugated proteins in cells. In addition to experiments examining the role of SUMO-1 conjugation in the regulation of RanGAP1 (see 1 Z01 HD008740-04), we have sought to identify conjugation targets whose modification is cell cycle-dependent. Our earlier results showed that Topoisomerase-II is modified exclusively by SUMO-2/3 during mitosis in Xenopus egg extracts, and that SUMO-2/3 conjugation may mobilize Topoisomerase-II from mitotic chromatin in a manner that is important for chromosome segregation at the metaphase-anaphase transition. In recent studies, we have found that the SUMO ligase PIASy is specifically required for mitotic conjugation of Topoisomerase-II to SUMO-2 in Xenopus egg extracts. PIASy was unique among the PIAS family members in its capacity to bind mitotic chromosomes and recruit the SUMO conjugating enzyme Ubc9 onto chromatin. These properties were essential, since PIASy mutants that did not bind chromatin or failed to recruit Ubc9 were functionally inactive. Moreover, PIASy depletion from extracts eliminated essentially all chromosomal accumulation of SUMO-2-conjugated species, suggesting that PIASy is the primary ligase for mitotic chromosomal substrates of SUMO-2. PIASy-dependent SUMO-2-conjugated species concentrated on the inner centromere, and inhibition of PIASy blocked anaphase sister chromatid segregation. Taken together, our observations suggest that PIASy is a critical regulator of mitotic SUMO-2 conjugation for Topoisomerase-II and other chromosomal substrates, and that its activity may have particular relevance for centromeric functions required for proper chromosome segregation.